Gene therapies hold the potential application in treating many genetic disorders. The success of gene therapies mainly depends on many different factors; one among them is the availability of regulable gene expression systems. The use of regulable gene expression systems is not only restricted to gene therapy applications; they are also useful for different functional genomic studies and clinical applications in mammals. As gene therapy research continuously progresses, the need for regulable gene expression systems becomes more apparent. An efficient regulable gene expression system should have the quality in controlling the level of expressed transgenes in a dose dependent manner in response to externally administered pharmacological agents. In addition, the regulable gene expression system should also have the ability in producing low level of background signal before administering the activators/regulators.
So far, several regulable gene expression systems have been developed and used for different applications. The very early systems include the naturally occurring physical and chemical stimuli responsive promoters such as heat shock, electric, light and heavy metal inducible promoters. Even though these natural promoters have the potential in controlling the level of transgene expression, adopting them for mammalian gene therapy application is difficult because of hazardous effects associated with the inducers. To overcome these issues, later combination elements derived from prokaryotic and eukaryotic systems were used for developing controlled gene expression systems. These systems are efficient for utilization in mammalian cells in vitro and in vivo. Most of these systems utilize either one or a combination of the following elements that includes DNA binding domains, ligand binding domains and transactivation domains. The systems developed by using these elements include tetracycline regulated system, mifepristone (RU486) regulated system, ecdysone regulated system, rapamycin regulated system, tamoxifen regulated system and ligand activated site specific recombination system (Cre-ER). Even though all these systems showed significant levels of transgene expression in response to externally administered pharmacological agents, many of them produced significant levels of background signal before administering the activators.
Estrogens are responsible for the growth, development and maintenance of many reproductive cells. The physiological effects of these hormones are mediated by a ligand-inducible nuclear transcription factor, the estrogen receptor (ER). In the classical pathway of steroid hormone action, 17β-estradiol binds to the ligand binding domain (LBD) of an estrogen receptor and induces homodimerization, which then binds to a specific regulatory sequence of promoters of ER target genes, the estrogen response elements (ERE). The binding of hormones and a variety of other chemicals to the LBD of ER leads to a series of molecular events. This includes the activation or repression of many downstream target genes through direct interaction with the transcription machinery.
Abnormal levels of estrogen have been linked with many disorders including cancer. The deficiency in the level of estrogen in post menopausal women can lead to reduced bone densities. Similarly, the presence of excess hormones has been reported to induce the development of different types of cancers including breast cancer. Most of these cancers respond to hormonal therapy (anti-estrogens) via the estrogen receptor. Hence, estrogen receptors are a major cellular therapeutic target.
The ER-LBD is folded into a three-layered, anti-parallel, α-helical sandwich composed of a central core layer of three helices that includes H5/6, H9, and H10. This is in turn sandwiched between two additional layers of helices (H1-4 and H7, H8, H11). This helical arrangement creates a “wedge shaped” molecular scaffold that maintains a sizeable ligand binding property at the narrower end of the domain. The remaining secondary structural elements, a small two-stranded, anti-parallel β-sheet (S1 and S2) and an α-helical H12, are located at this ligand binding portion of the molecule and flank the three-layered motif. The helix 12 (H12) is mainly located in the pocket of the ligand binding region. Therefore, it is a key element of the receptor in developing conformational modifications in response to various ligands. The crystal structures of the LBD complexed with 17β-estradiol and Raloxifene show that although both ligands bind at the same site within the core of the LBD, each of these ligands induces a different conformational change on H12. In addition, the binding of ligands to the ligand-binding domain of ERα causes a conformational shift of helix 12 into an adjacent co-activator site that either prevents or enhances ERα from binding to a co-activator (NR box peptide), which would then activate a specific DNA sequence, the estrogen response element (ERE). This process controls many genes that are responsible for cell growth. Hence, helix 12 is one of the major portions of ER that plays a critical role in the ligand induced proliferative effect of cells, and it is therefore important to develop an assay based on the movement of helix 12 in response to different ligands.